Sequence-specific binding of proteins to DNA, RNA, protein and other molecules is involved in a number of cellular processes such as, for example, transcription, replication, chromatin structure, recombination, DNA repair, RNA processing and translation. The binding specificity of cellular binding proteins that participate in protein-DNA, protein-RNA and protein-protein interactions contributes to development, differentiation and homeostasis. Alterations in specific protein interactions can be involved in various types of pathologies such as, for example, cancer, cardiovascular disease and infection.
Zinc finger proteins (ZFPs) are proteins that can bind to DNA in a sequence-specific manner. Zinc fingers were first identified in the transcription factor TFIIIA from the oocytes of the African clawed toad, Xenopus laevis. A single zinc finger domain of this class of ZFPs is about 30 amino acids in length, and several structural studies have demonstrated that it contains a beta turn (containing the two invariant cysteine residues) and an alpha helix (containing the two invariant histidine residues), which are held in a particular conformation through coordination of a zinc atom by the two cysteines and the two histidines. This class of ZFPs is also known as C2H2 ZFPs. Additional classes of ZFPs have also been suggested. (See, e.g., Jiang et al. (1996) J. Biol. Chem. 271:10723-10730 for a discussion of Cys-Cys-His-Cys (C3H) ZPFs.) To date, over 10,000 zinc finger sequences have been identified in several thousand known or putative transcription factors. Zinc finger domains are involved not only in DNA recognition, but also in RNA binding and in protein-protein binding. Current estimates are that this class of molecules will constitute about 2% of all human genes.
Most zinc finger proteins have conserved cysteine and histidine residues that tetrahedrally-coordinate the single zinc atom in each finger domain. In particular, most ZFPs are characterized by finger components of the general sequence: -Cys-(X)2-4-Cys-(X)12-His-(X)3-5-His (SEQ ID NO: 1), where X is any amino acid (the C2H2 ZFPs). The zinc-coordinating sequences of this most widely represented class contain two cysteines and two histidines with particular spacings, for example zinc fingers found in the yeast protein ADRI, the human male associated protein ZFY, the HIV enhancer protein and the Xenopus protein Xfin have been solved by high resolution NMR methods (Kochoyan, et al., Biochemistry, 30:3371-3386, 1991; Omichinski, et al., Biochemistry, 29:9324-9334, 1990; Lee, et al., Science, 245:635-637, 1989). Based on x-ray crystallography, the three-dimensional structure of a three finger polypeptide-DNA complex derived from the mouse immediate early protein zif268 (also known as Krox-24) has been solved. (Pavletich and Pabo, Science, 252:809-817, 1991). The folded structure of each finger contains an antiparallel β-turn, a finger tip region and a short amphipathic α-helix. The metal coordinating ligands bind to the Zn ion and, in the case of zif268 zinc fingers, the short amphipathic α-helix binds in the major groove of DNA. In addition, the conserved hydrophobic amino acids and zinc coordination by the cysteine and histidine residues stabilize the structure of the individual finger domain.
The folding of a C2H2 ZFP into the proper finger structure can be entirely disrupted by exchange of the C2H2 ligand amino acids. Miura et al. (1998) Biochim. Biophys. Acta 1384:171-179. Furthermore, metal binding specificity of peptides based on the C2H2 consensus sequence can be altered. Krizek et al. (1993) Inorg. Chem. 32:937-940; Merkle et al. (1991) J. Am Chem. Soc. 113:5450-5451. Although detailed models for the interaction of zinc fingers and DNA have also been proposed (Berg, 1988; Berg, 1990; Churchill, et al., 1990), mutations in finger 2 of the three-fingered C2H2 ZFP zif268 have been shown to entirely abolish DNA binding (Green et al. (1998) Biochem J. 333:85-90).
Nonetheless, increased understanding of the nature and mechanism of protein binding specificity has encouraged the hope that specificity of a binding protein could be altered in a predictable fashion, or that a binding protein of predetermined specificity could be constructed de novo. See, for example, Blackburn (2000) Curr. Opin. Struct. Biol. 10:399-400; Segal et al. (2000) Curr. Opin. Chem. Biol. 4:34-39. To this end, attempts have been made to modify C2H2 zinc finger proteins. See, e.g., U.S. Pat. Nos. 6,007,988; 6,013,453; 6,140,081; PCT WO98/53057; PCT WO98/53058; PCT WO98/53059; PCT WO98/53060; PCT WO00/23464; PCT WO 00/42219; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; Segal et al. (2000) Curr. Opin. Chem. Biol. 4:34-39; and references cited in these publications.
To date, however, cellular studies using designed C2H2 ZFPs have utilized relatively few positions in the zinc finger as adjustable parameters to obtain optimal activity. In particular, studies to date have modified only those residues at the finger—DNA interface. These have included positions known to make direct base contacts, ‘supporting’ or ‘buttressing’ residues immediately adjacent to the base-contacting positions, and positions capable of contacting the phosphate backbone of the DNA. Furthermore, many observed effects have been quite modest, and the possibility that improved ZFP activities might be achieved via substitution of residues at other positions in the finger or using non-C2H2 polypeptides has remained completely uninvestigated.
Thus, there exists a need for additional designed or selected zinc finger binding proteins.